Non-alcoholic fatty liver disease (NAFLD) is the most frequent liver disease in the US, affecting approximately a third of the US adult population. Obesity is the most common risk factor for NAFLD. NAFLD encompasses a spectrum from simple fatty liver to non-alcoholic steatohepatitis (NASH). Advanced NASH includes fibrosis and can progress to cirrhosis and hepatocellular carcinoma. The factors that promote the progression to NASH and fibrosis are not well known. It is widely accepted that this is caused by a second hit, but the nature of this second hit remains elusive. It is well established that obesity causes an increase in the systemic levels of lipopolysaccharide (LPS). Additionally, we demonstrated that obesity increases levels of the damage- associated molecular pattern high mobility group box 1 (HMGB1) in mice and patients. Both LPS and HMGB1 trigger activation of Toll-like receptor 4 (TLR4) signaling, a pathway with major contributions to hepatic inflammation and fibrosis. Based on these data, we hypothesize that the progression of simple hepatic steatosis to NASH and fibrosis is triggered by activation of the TLR4 pathway by LPS and/or HMGB1. We seek to test this hypothesis by (1) examining whether TLR4 contributes to obesity-induced liver fibrosis, and (2) by determining the TLR4 ligands that drive progression in obesity-induced liver disease. To achieve these aims, we will use a model of diet-induced obesity that includes a high-fat, high-saturated fat diet and administration of fructose in the drinking water, mimicking a fast food western-style diet (FFD diet). The FFD model has major advantages over the widely used methionine-choline-deficient model which typically leads to severe weight loss, and thus does not model human obesity and NAFLD. In contrast, FFD diet causes obesity, liver steatosis, NASH, and fibrosis and therefore is a physiological representation of human NAFLD. To study the role of TLR4, we will use mice with global and tissue-specific deletions of TLR4, including a novel and highly efficient method to selectively delete TLR4 in hepatic stellate cells. We will evaluate whether the deletion of TLR4 prevents hepatic inflammation and fibrosis. To study the role of LPS and HMGB1, we will evaluate whether pharmacological and genetic strategies that suppress LPS or HMGB1-mediated activation of TLR4 prevent obesity-driven hepatic inflammation and fibrosis. Results from these studies will improve our understanding of the mechanism that trigger disease progression in NAFLD and may point to new and clinically feasible therapeutic strategies.